Midterm 1 Flashcards

Question

FRET - What is it?

Answer 1

Fluorescence Resonance Energy Transfer Technique used to study the dynamics of molecular mechanisms (like protein interactions, folding dynamics or conformational chnages) through the transfer of fluoresence between a donor and acceptor.

Answer 2

The Forster distance of 1-10 nm

Answer 3

1. The Donor fluorophore is excited at a particular wavelength and absorbs a photon. 2. The acceptor fluorophore will be transferred energy when <10nm away. 3. The acceptor will emit light at a longer wavelength from the donor and FRET efficency will be measured.

Answer 4

Because of its sensitivity to nanometer-scale distance changes, FRET is a powerful tool for studying dynamic biomolecular processes in real time.

Answer 5

A technique used to measure rotational motion of fluorescent molecules. Fluorophore is excited with polarized light and loses polarization depending on the size of the molecule/rotation speed before excitation occured. - Small molecules rotate fast and leads to low fluorescence polarization. - Large molecules rotate slowly and retain polarization giving a high fluoresence polarization. Applications: Protein-Ligand binding, enzyme activity, antibody-antigen activity and DNA-protein interactions.

Answer 6

Circular Dichroism is a technique used to study chiral properties of molecules through the difference in absorption between left and right-handed ciruclar polarized light. - It is primarily used to determine secondary stuctures.

Answer 7

Empirical rules and statistical parameters to predict secondary structures. Used in simple cases for CD.

Answer 8

- Determine secondary structures - Investigate drug binding - Study the dynamics of protein folding - Ligand induced conformational changes - protein-protein interactions and protein nucleic acid interactions.

Answer 9

Surface Plasmon Resonance Observe the interaction of macromolecules (like proteins, nucleic acids, lipid vesicles, virsues, etc.) on the surface of a sensor in real time without the need for a label.

Answer 10

- Affinity (binding strength) - Kinetics (Speed of formation/dissociation) - Concentration - Specificity (For its target) - Comparability (to reference products)

Answer 11

1. Salting out 2. Isoelectric precipitation 3. Precipitation through organic solvets

Answer 12

1. Ion Exchange 2. Affinity 3. Size-Exclusive 4. Hydrophobic Interaction 5. Isoelectric focusing

Answer 13

Seperates proteins based on charge. The matrix can either be positively charged to sort our negatively charged proteins (Cation exchange) or negatively charged to seperate out positively charged proteins (anion exchnage).

Answer 14

Seperates proteins based on binding interactions. A mixture of proteins are added to a column followed by a specific ligand. the ligand will bind to one type of protein and the rest will elute quickly.

Answer 15

1. pH - Change in pH can disrupt binding. 2. Ion Strength - Adding salt weakens electrostatic interaction to release protein from ligand. 3. Denature - Use of an agent to disrupt H-bonding. 4. Competitor ligand - Compettive molecule displaces bound protein on the ligand.

Answer 16

Seperates prtoeins based on size and molecular weight. The column is filled with carbohydrate polymer beads. smaller molecules will enter the pores of the beads, slowing their movement whereas larger molecules bigger than the beads will pass through the column quickly.

Answer 17

Seperates proteins based on hydrophobicity. Proteins are loaded onto a column in a high salt concentration. Proteins with higher hydrophobicity bind more strongly to the column's hydrophobic ligands.

Answer 18

Seperates proteins based on their isoelectric point (pI). Similar to ion-exchange, but it is the change in buffer pH (decreasing) causes the proteins to elute in order of pI.

Answer 19

1. Dialysis 2. Ultrafiltration 3. SEC 4. Centrifugation 5. Electrophoresis

Answer 20

Uses a semi-permeable membrane to separate proteins based on molecular size. Small molecules diffuse out, while larger proteins remain inside the dialysis bag.

Answer 21

Similar to dyalisis, but doesn't use the principle of diffusion. Instead the smaller proteins are passed through the semipermeable membrane due to applied pressure. The pores in the semipermeable membrane can have modified MWCO (molecular weight cut-offs).

Answer 22

Separates proteins based on density and size using high-speed spinning

Answer 23

Differential centrifugation - Separates by sedimentation rate (SIZE) Density gradient centrifugation - Separates by buoyant density (DENSITY)

Answer 24

Separate proteins on the basis of their size, shape or charge. Uses an electric field

Answer 25

Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis Separates proteins based on molecular weight under denaturing conditions. Smaller proteins migrate faster than larger proteins due to the detergent, SDS. It will bind to every other amino acid, therefore giving a negative charge propotional to its size.

Answer 26

It is a cross-linking reagent used in the polacylamide gel or SDS-PAGE, allowing for the variation in pore size.

Answer 27

Separates proteins based on their isoelectric point (pI). Proteins migrate in a pH gradient until they reach a pH where their net charge is zero.

Answer 28

Combines IEF (separates by pI) and SDS-PAGE (separates by size).

Answer 29

Used to detect specific proteins after SDS-PAGE. 1. Protein seperation via SDS-PaGE. 2. Transfer to a membrane 3. Add a blocking agent to the membrane to prevent non-specific binding of antibodies. 4. Addition of primary antibody that binds to target protein. 5. Addition of secondary antibody with a detection enzyme. 6. Signal detection via the addition of substrate or visualize with fluorescence.